L-ornithine, the intermediate of L-arginine biosynthesis, has already been widely used as a dietary supplement, as it is known to be beneficial for the treatment of wound healing and liver disease. Furthermore, it is also the precursor of bulk chemicals such as putrescine, an important diamine used as a nylon monomer, and natural products such as tropane alkaloids, which are used as parasympatholytics for competitively antagonizing acetylcholine.
L-ornithine is nowadays prepared by various processes, encompassing chemical synthesis and enzymatic catalysis. For instance, EP0464325A2 discloses an enzymic conversion process for the preparation of salts of L-ornithine from L-arginine in the presence of the enzyme L-arginase (EC 3.5.3.1.) extracted from animal liver. To reduce the cost of the enzyme, a whole-cell biotransformation system for the conversion of L-arginine to L-ornithine was also developed by constructing a recombinant Escherichia coli with overexpressed arginase (EC 3.5.3.1) encoding gene ARG from the bovine liver (Zhan et al. 2013). However, said whole-cell biotransformation system always has the problem of cell permeability, and addition of permeability reagent may lead to subsequent product separation problems. One idea has been to screen for a thermophilic enzyme, higher operation temperature could be used to improve the permeability of the recombinant cells. Arginase (ARG) from Bacillus caldovelox was found to be a potential thermophilic candidate (Patchett et al. 1991). Recently, the recombinant E. coli with B. caldovelox ARG gene was constructed, leading to an efficient and simple enzymatic process for the environment-friendly synthesis of L-ornithine from L-arginine (Song et al. 2014).
However, these methods either suffer from issues of expensive substrates, poor enantiopure purity or are environmental unfriendly.
Some L-citrulline or L-arginine auxotroph bacteria belong to the genus Brevibacterium, the genus Corynebacterium, the genus Bacillus and the genus Arthrobacter are known to produce L-ornithine. In addition, variants of said L-ornithine producing bacteria having resistance to arginine analogues and/or 2-thiazolealanine and/or sulfaguanidine and/or 2-fluoropyruvic acid and/or microphenolic acid and/or ornithinol are said to have better L-ornithine producing performance. Metabolic engineering frameworks, which offer the ability to leverage the advantages of biocatalysts (e.g. precision, specificity) and tailor the carbon flow of microbes, have enabled construction of platform cell factories for producing amino acids. Recent advantages in the said metabolic engineering also lead to the strain construction for L-ornithine production (Hwang and Cho 2012; Hwang and Cho 2014; Hwang et al. 2008; Jiang et al. 2013a; Jiang et al. 2013b). For instance, WO 2012008809 A2 discloses a strategy to give the L-ornithine production at a high yield rate and with high efficiency by the fine-turning of gluconate kinase (GntK) in Corynebacterium. In another disclose, U.S. Pat. No. 8,741,608 B2 suggests that overexpressing of L-ornithine exporter is an efficient strategy to further improve L-ornithine titers. US 20140051132 A1 also discloses an invention where NCgl_2067-NCgl_2065 operon in Corynebacterium is/are attenuated lead to the improved production of L-amino acids belonging to L-glutamate family which includes L-ornithine. NCgl_2067-NCgl_2065 operon was suggested to encode negative regulators which directly controls expression of the related genes in the L-amino acids synthesis belong to L-glutamate.
However, one of the main drawbacks of L-ornithine production in bacterial strains is the phage contamination issues which often result in substantial economic losses. Thus, there is a need for better microbial strategies for the production of L-ornithine, putrescine, spermidine, spermine and other chemicals using these compounds as a precursor